Development and optimization of lipoplex vectors for an antisens therapeutic approach in the context of HPV induced lesions

Development and optimization of lipoplex vectors for an

Development and optimization of lipoplex vectors for an

antisens therapeutic approach in the context of HPV induced

antisens therapeutic approach in the context of HPV induced

lesions

lesions

Contact : anna.lechanteur@student.ulg.ac.be

Anna Lechanteur1_{,Brigitte Evrard}1_{,Philippe Delvenne}2_{, Patrick Roncarat}2 _{,Pascale Hubert}2_{, Geraldine Piel}1 1_{Laboratory of Pharmaceutcal Technology-CIRM,} 2_{Laboratory of Experimental Pathology, GIGA-CANCER}

University of Liege, Liège, Belgium

HPV 16 and 18 are responsible for cervical cancers, in over 70% of cases. These viruses integrate into keratnocyte cells and induce

the expression of oncogenes E6 and E7. These prevent the expression of tumor suppressor genes (p53 and pRb) and lead

keratnocytes transformaton into tumor cells.

The purpose of this study is to target locally mRNA encoding for E6-E7 oncoproteins with siRNA. In order to protect and to

optmize their penetraton through the vaginal mucus and into the cytoplasm, siRNA will be incorporated into catonic liposomes.

HPV 16 and 18 are responsible for cervical cancers, in over 70% of cases. These viruses integrate into keratnocyte cells and induce

the expression of oncogenes E6 and E7. These prevent the expression of tumor suppressor genes (p53 and pRb) and lead

keratnocytes transformaton into tumor cells.

The purpose of this study is to target locally mRNA encoding for E6-E7 oncoproteins with siRNA. In order to protect and to

optmize their penetraton through the vaginal mucus and into the cytoplasm, siRNA will be incorporated into catonic liposomes.

Cervical cancer

 Human Papillomavirus responsible for cervical cancer

Histological development of cervical cancer Development of cervical cancer

Frequently two (DOTAP and DOTMA)

Biodegradable (DOTAP = ester) Essential CATIONIC charged (but toxic)

Liposomes

Cationic lipids used: general structure

Lipid bilayer Internal aqueous compartment



Characteristics required

• High transfecton

• Selectve for E6 and E7 proteins • Induce APOPTOSIS of cancer cells



Design rules

• Double strand (17-25 nucleotdes) • + 2dTdT at the 3’end of the antsens sequence • GC content less than 50%

• …

siRNA

2 sequences selected

: - siRNA targetng E6 (against HPV16) :

- siRNA targetng E7 (against HPV16) :

1) Cells transfecton using

Transfectne ®

• HPV16+ : SiHa, CaSki • HPV16- : C33A • HPV18+ : C4 II

2) Tests

• % transfecton (FACS)

• % decrease of oncoproteins E6 and E7 (QRT-PCR)

• % apoptosis: Annexin V-PI (FACS)

5’---CUAGGCAAACAACUAUACAUGAUAdTdT---3’

3’---dTdTGAUCCGUUUGUUGAUAUGUACUAU---5’

5’---AGGAGGAUGAAAUAGAUGGdTdT---3’

3’---dTdTUCCUCCUACUUUAUCUACC---5’

Physico-chemical characterization:

Morphology, size (<200nm), zeta potental (0-40mV), physical stability over the tme, encapsulaton rate, capacity to release their content,…

Tests on cells:

• HPV16+ : SiHa, CaSki • HPV16- : C33A • HPV18+ : C4-II

 Tests on organotypic cultures to evaluate penetration efficiency

Validation of the concept in vivo (mice)

Tools to improve the efficiency of lipoplexes

• Add neutral lipids (DOPE, cholesterol), PEG, change proportons of compounds,…

Lipoplexes

Evaluaton of the transfecton efficiency and cytotoxicity